Low-Density Lipoprotein (LDL)-Antioxidant Biflavonoids from Garcinia madruno

Six biflavonoids were isolated from G. madruno, one of which, 7''-O-(6''''-acetyl)-glucoside of morelloflavone, is a new compound identified on the basis of 1D, 2D NMR (HMQC and HMBC) spectroscopic methods and chemical evidence. The antioxidant activity of the biflavonoids against low-density lipoprotein (LDL) peroxidation induced with Cu2+, was studied by means of a TBARS assay. The antioxidant potential of a biflavonoid fraction (BF) was also evaluated and correlated with its biflavonoid content. The flavanone-(3→8'')-flavone biflavonoids displayed antioxidant activity, particularly morelloflavone, which was significantly more potent than quercetin, with a CE50 of 12.36 μg/mL. Lipid peroxidation, was also significantly reduced in the presence of the BF (EC50 = 11.85 μg/mL). These results suggest that the BF is an excellent antioxidant.


Introduction
Garcinia madruno (Kunth) Hammel, commonly known as madroño, is a tree endemic to Central and South America.It is resistant to plagues and illnesses and adaptive to different environmental conditions [1].Extracts obtained from G. madruno show antibacterial activity and are particularly OPEN ACCESS efficient against Staphylococcus aureus [2].Other species of the Garcinia genus have been reported to exhibit diverse biological properties, such as anti-inflammatory, antioxidant, antiimmunosuppressive, antitumor promoter, cytotoxicity, antinematodal, antiviral, antiplasmodial, trypanocidal, and antimicrobial activity, and also in healing skin infections and wounds [3][4][5][6][7][8][9][10]. Phytochemical studies of this genus have revealed the presence of xanthones, benzophenones and biflavonoids [3,[11][12][13].Consequently, we became interested in carrying out a comprehensive investigation of the twigs and leaves of G. madruno.In a previous investigation, we report the inhibitory LDL oxidation potential and free radical stabilization capacity of a biflavonoid fraction (FB) from G. madruno [14].This paper deals with the isolation and characterization of a new biflavonoid, along with five known biflavonoids.The relative antioxidant activity of the biflavonoids against LDL peroxidation is also reported.

Biological Activity
All compounds and the BF were tested for their antioxidant activity against LDL-peroxidation (Figure 3).The flavanone-(3→8'')-flavone biflavonoids displayed antioxidant activity, particularly compound 2, which was significantly more potent than quercetin, with a CE 50 of 12.36 μg/mL (p-value < 0.05).The antioxidant potential of the BF was evaluated and correlated with its biflavonoid content, which was identified as amentoflavone (1), morelloflavone (2) and volkensiflavone (3).Cu 2+ -induced LDL oxidation was significantly reduced in the presence of the BF (EC 50 = 11.85 μg/mL), mainly due to biflavonoid 2, although synergy processes might also be involved.In fact, it has been reported that kolaviron, a biflavonoid fraction composed by GB-1, GB-B2 and kolaflavanone, increases lipoprotein resistance to copper-induced oxidation in rats, and also, in vitro, it protects against Cu 2+ -induced oxidation of rat serum lipoprotein, presumably by mechanisms involving metal chelation and antioxidant activity [29].

Plant Material
The aerial parts of G. madruno were collected in Medellí n (Colombia).This sample was identified by M.Sc.Fernando Alzate.A voucher specimen (Alz-3030) has been deposited at the Herbarium of Universidad de Antioquia (HUA).

Inhibition of LDL Oxidation
The protective effect of the biflavonoids and the BF against LDL-peroxidation was determined by a TBARS assay.
3.5.1.Human LDL Isolation 50 mL of blood was collected by venepuncture into heparinized tubes from healthy non-smoking volunteers (20-25 years old).Plasma was recovered by differential density ultracentrifugation at 2,500 rpm and 4 °C, in a Beckman XL-100 ultracentrifuge (Brea, CA, USA) equipped with a SW-55Ti rotor, as described elsewhere.The LDL fraction was obtained by centrifugation with 1.6 mL of NaCl (17 M) in distilled water at 49,500 rpm for 12 h.The superior fraction was removed and 1.6 mL of KBr (10 M) was added before another centrifugation for a period of 18 h.SDS-PAGE was used to confirm the purity of the collected fractions (kilomicrons, VLDL, LDL and HDL).The concentration of protein was determined by the Protein Quantification Kit-Rapid method of Fluka ® (St.Louis, MO, USA).

TBARS Determination
The formation of products from peroxidation of LDL was determined by the thiobarbituric acid reactive substances assay (TBARS).The LDL was incubated at 37 °C in 0.1 M potassium phosphate buffer, and made up to a final protein concentration of 300 μg/mL.Volumes of 50 μL of biflavonoids or quercetin (positive control) at different concentration were added and the peroxidation was initiated by 50 μL of CuSO 4 100 μM, and finished by 5 μL of 1% EDTA and cooling.A TCA-TBA-HCl stock solution (15% w/v trichloroacetic acid; 0.67% w/v thiobarbituric acid; 0.1N HCl) was added to the reaction mixture.The solution was then heated at 95 °C for 60 min.The supernatant was filtered through a 0.45 μm membrane and a reading was made at 532 nm.Readings of three independent experiments were carried out.One-way ANOVA with a Student Newman-Keuls post-test was performed using GraphPad Prism version 4.00 and p < 0.05 was considered as significant difference.

Conclusions
In summary, six biflavonoids were isolated from G. madruno.Compound 5 was found to be a new biflavonoid glycoside on the basis of spectroscopic analyses and chemical evidence.Some of the compounds exhibited potent lipid peroxidation inhibition activity, and lipid peroxidation was also significantly reduced in the presence of the biflavonoid fraction (BF), mainly due to morelloflavone.These results suggest that the BF is an excellent antioxidant.